For cell types not amenable to lipid-mediated transfection, viral vectors are often employed. Virus-mediated transfection, also known as transduction, offers a means to reach hard-to-transfect cell types for protein overexpression or knockdown, and it is the most commonly used method in clinical research (Glover et al., 2005; Pfeifer and Verma, 2001). Adenoviral, oncoretroviral, and lentiviral vectors have been used extensively for gene delivery in mammalian cell culture and in vivo. Other well-known examples for viral gene transfer include baculovirus and vaccinia virus-based vectors. For the more information on various viral delivery systems, see Viral Vectors.
While viruses are the preferred system for gene delivery in clinical trials owing to their high in vivotransfection efficiency and sustained gene expression due to their integration into the host genome, they have a number of drawbacks including their immunogenicity and cytotoxicity, technically challenging and laborious production procedures for vectors, high costs due to biosafety requirements, low packaging capacity (~10 kb for most viral vectors compared to ~100 kb for non-viral vectors), and variability in the infectivity of viral vector preparations (Glover et al., 2005; Kim and Eberwine, 2010; Vorburger and Hunt, 2002).
A typical transduction protocol involves engineering of the recombinant virus carrying the transgene, amplification of recombinant viral particles in a packaging cell line, purification and titration of amplified viral particles, and subsequent infection of the cells of interest. While the achieved transduction efficiencies in primary cells and cell lines are quite high (~90–100%), only cells carrying the viral-specific receptor can be infected by the virus. It is also important to note that the packaging cell line used for viral amplification needs to be transfected with a non-viral transfection method.
Generate recombinant virus via gene cloning.
Transfect packaging cell line using a non-viral method to amplify and isolate viral vector.
Purify and titrate the viral vector containing the transgene.
Infect cells of interest (containing viral-specific receptor) at appropriate multiplicity of infection (MOI).
Remove virus from the culture and/or add fresh medium.
Assay transduced cells for gene expression or silencing.
Viral delivery workflow.